Separation of liquid in droplets and sedimented material enclosed therein

ABSTRACT

The invention relates to methods for drawing-off liquid from individual droplets which are in a predefined arrangement on a flat substrate and have sedimented material enclosed in them. A mask of an absorbent material comprising a pattern of indentations or holes which corresponds at least partially to the regular arrangement of the individual droplets, or a stiff, rigid plate of an absorbent material is positioned above the flat substrate in such a way that the droplets come into contact with the absorbent material peripherally so that liquid is drawn off there-into. The invention also relates to a mask of an absorbent material with a substantially rectangular shape which has a predefined pattern of indentations or holes for the purpose of separating liquid and sedimented material enclosed therein.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to methods for drawing-off liquid from individualdroplets which are in a predefined (especially regular) arrangement on aflat substrate and have sedimented material enclosed in them. Theinvention also relates to a mask of an absorbent material with asubstantially rectangular shape which has a predefined (regular) patternof indentations or holes for the purpose of separating liquid andsedimented material enclosed therein.

Description of the Related Art

There is a need to gently separate liquid from the sedimented materialenclosed in it so that as little as possible of the sedimented materialis removed together with the liquid in this separation process, whileunsedimented (i.e. still suspended) constituents are also to be drawnoff with the liquid.

The Prior Art is explained below with reference to a special aspect.This shall not be understood as a limitation, however. Useful furtherdevelopments and modifications of what is known from the Prior Art canalso be used above and beyond the comparatively narrow scope of thisintroduction, and will easily be evident to the expert skilled in theart in this field after reading the following disclosure.

Earlier tests have shown that microorganisms which are suspended in adroplet of nutrient liquid on a flat substrate accumulate in a sedimentof microorganisms after a relatively short standing time (or “resttime”) of up to one hour. The microorganisms which are sedimented therein a kind of “biofilm” can be carefully separated from the residualliquid and the remaining suspended particulate matter, by bringing anabsorbent cloth into contact with the droplets, for example. After this“dehydration”, the species of the microorganisms can be reliablydetermined with a subsequent mass spectrometric measurement, cf.international application PCT/DE2016/100561. This finding wasastonishing because, contrary to expectation, it was found that the cellsediment of the microorganisms of interest was not removed together withthe drawn off liquid. This finding allows the cultivation (orincubation) of microorganisms to promote growth and the preparation foran analytical measurement on one and the same substrate, such as asample support plate for inserting into the ion source of a massspectrometer.

As yet there is no fully developed scientific explanation for thismicrobial behavior in a droplet on a flat substrate. It is assumed,however, that physical interactions between the plate surface and thecells of the microorganisms, and adhesion processes caused by thebiochemical and biophysical properties of the cell surface of themicroorganisms, are responsible for the preferred attachment orsedimentation on the substrate.

Further objectives to be achieved by the invention will suggestthemselves immediately to the person skilled in the art after readingthe disclosure below.

SUMMARY OF THE INVENTION

The invention relates generally to methods and devices for drawing offliquid from individual droplets which are in a predefined (especiallyregular) arrangement on a flat substrate and have sedimented materialenclosed in them.

In accordance with a first aspect, the invention relates to a methodwherein a mask of an absorbent material which preferably has a patternof indentations or holes corresponding at least in part to thepredefined arrangement of the individual droplets, is positioned abovethe flat substrate in such a way that the center of the indentations orholes and the center of the droplets are substantially above one anotherin each case. The edges of the indentations or holes come into contactwith peripheral parts of the individual droplets in each case, while thesedimented material remains untouched, and liquid is thus drawn off intothe absorbent material.

A great advantage compared to the Prior Art is that the liquid fromlarge numbers of (equal) droplets can be removed simultaneously with amask of an absorbent material. As a flat substrate, a sample supportplate can be coated with liquid droplets at several or even all samplespots, for example, and by applying the mask with the pattern ofindentations or holes, the sedimented particles in all droplets can beseparated simultaneously from the liquid, which would otherwiseinterfere with the further processing. Furthermore, the design of themask with indentations or holes whose dimensions are adapted in thebroadest sense to the size of the droplets to be absorbed, ensures thatthe liquid can be drawn off simultaneously over the whole circumferenceof the droplet (360°), which accelerates the process and optimizes theutilization of the liquid absorbency of the mask material.

A further advantage is that—unlike a plate or cloth of absorbentmaterial without indentations or holes—the vertical separation betweenthe absorbent material of the mask and the flat substrate can be chosento be more or less as desired. It is even possible to choose thepressure with which the mask is applied to the flat substrate more orless as desired. This increases the robustness of the handlingconsiderably. Other embodiments of the method using rigid, stiff platesof absorbent material, without being necessarily profiled withindentations or perforated with holes, will be described further below.

The droplets may, in particular, mainly contain liquid which is used insample preparation methods and sample processing methods for infraredspectroscopy or mass spectrometry. The droplets may contain nutrientliquid, for example, and the sedimented material may comprisemicroorganisms cultivated in this droplet of nutrient liquid and thenprecipitated. Separating the nutrient liquid from sedimentedmicroorganisms can, in particular, serve as a processing step of asample for infrared-spectroscopic or mass-spectrometric identification(e.g. by means of IR transmission spectroscopy or MALDI time-of-flightmass spectrometry) according to species/subspecies, or othercharacterization of the microorganisms, such as the rapid determinationof resistance/sensitivity of the microorganisms against antimicrobialsubstances.

The droplets may additionally or alternatively contain a washing liquidused for a sample processing procedure (for example an aqueous solutionor pure de-ionized water) or other liquid processing media. For example,the washing liquid from sample spots which have been coated with apreviously dried sample or with dried matrix substance and sample can bedrawn off with the mask. The washing liquid is preferably chosen suchthat the matrix substance or the matrix crystal lattice with embeddedsample crystals is not dissolved, e.g. with an α-cyano-4-hydroxycinnamicacid affinity preparation for in-situ desalting (cf. Gobom et al., Anal.Chem. 73, 2001, 434-438).

In various embodiments, a metal or ceramic plate can be used as the flatsubstrate. Possible plates are, particularly, polished stainless-steelplates or modifications thereof, such as the so-called anchor plates(AnchorChip™; Bruker Daltonik GmbH), which contain sequences ofalternate, delimited lyophilic and lyophobic areas on a stainless steelsubstrate. The plate may be reusable or disposable.

In spectroscopic or mass spectrometric analysis in particular, 48, 96,384 or 1536 individual droplets can be arranged in a predefined (andregular) pattern on the flat substrate. The sedimented material enclosedin the individual droplets may contain microorganisms, for example.

Usual droplet volumes can be approximately one to twelve microliters.These volumes in uniformly shaped droplets correspond roughly to adiameter of two to three millimeters above the flat substrate. It is ofcourse also possible to design the flat substrate with preferred dropletspots, for example lyophilic circular areas in lyophobic surroundings.The diameter of the droplets will then adjust itself to match thedimension of the lyophilic circular area, as happens with standardizedanchor plates of the AnchorChip™ type.

In various embodiments, the mask and the flat substrate can be movedslightly relative to each other (indicated as a slight horizontal wipingmovement) to ensure all the individual droplets come into contact withthe corresponding indentations or hole edges and thus ensure efficientremoval (e.g. to take into account a lack of precision when applying thedroplets). As soon as contact is made, the liquid is drawn off from thedroplet in a very short time by means of the capillary forces of theabsorbent material; the sedimented material, on the other hand, remainsat the site on the flat substrate and can be further treated orprocessed.

An average droplet diameter can preferably be slightly larger than thediameter of an indentation or hole. If different droplet volumes areused for different procedures, it is possible to provide correspondingmasks which are ready made in different sizes. In cases where thedroplets are arranged on particularly hydrophilic patches in hydrophobicsurroundings (e.g. AnchorChip™ plates), the diameters of theindentations or holes are correspondingly slightly smaller than theanchor patches.

A frame for the mask or a vertical guide for the flat substrate, intowhich the mask is inserted, can be provided and assists in guiding andaligning the mask as it is lowered onto the droplet array. The frame canbe fixed to the mask, for example, by folding a projecting, custom-cutedge of the absorbent material and then impregnating it with a plasticmaterial which then sets. The frame can also be attached to the outercircumference of the mask using an injection molded plastic.

In various embodiments of the methods, the mask may consist of a thick,flexible cloth of absorbent material being laterally mounted tensely ina surrounding holder frame (membrane-like).

According to a further embodiment, a rigid, stiff (and flat) plate of anabsorbent material is positioned above the flat substrate using a spacerridge, which is located laterally to the predefined arrangement, in sucha way as to prevent the absorbent material and the flat substrate fromestablishing contact while facilitating protruding parts of theindividual droplets coming into contact with the absorbent material as aresult of which liquid is drawn off there-into. The spacer ridge may bedesigned and configured to keep a distance of about one third to abouthalf the droplet diameter above the flat substrate surface, forinstance. Primary advantages of this variant are that there is (i) nospecial alignment of the predefined arrangement and the rigid, stiffplate required and (ii) hardly any wear and tear of the flat substratesurface carrying the droplets since it works completely without physicalcontact.

It goes without saying that all features and implementations set outpreviously in the context of the first aspect do equally apply to thisvariant, if practicable.

By using the rigid, stiff plate and the spacer ridge, the sedimentedparticles in all droplets can be separated simultaneously from theliquid, which would otherwise interfere with the further processing. Assoon as contact is made, the liquid is drawn off from the droplet in avery short time by means of the capillary forces of the absorbentmaterial; the sedimented material, on the other hand, remains at thesite on the flat substrate (untouched) and can be further treated orprocessed.

A flat substrate receptacle may be provided in which the flat substrateis accommodated and which comprises the spacer ridge, such as a ledgerthat surrounds the predefined arrangement over the full periphery(360°). Additionally or alternatively, the spacer ridge may be locatedat the plate surface near the plate edges facing the flat substrate,which would ease the design requirements for a receptacle, for instance.If different droplet volumes are used for different procedures, by wayof example, it is possible to provide the rigid, stiff plates withcorresponding spacer ridges which are ready made in different sizes.

In accordance with a second aspect, the invention relates to a mask ofabsorbent material of substantially rectangular shape which has apredefined (especially regular) pattern of indentations or holes.

The mask is preferably manufactured from a stiff, rigid material. It canbe manufactured from profiled or perforated filter paper, non-wovenmaterial or cardboard, for example. Rigid materials are particularlysuitable for automated procedures for drawing off liquid. Usingspecially adapted handling devices such as robot arms, the rigid maskscan easily be removed from a storage place, clamped into an adaptedholder, conveyed to the liquid removal stage and then set down in therequired place, and disposed of, if required.

To produce the masks, it is possible to take a length of the absorbentmaterial, cut off pieces of the mask of a size to suit the desired outercontour of the mask, and then press or cut the predefined pattern ofindentations or holes into them, the latter by punching, for example.Particularly if it is to be used in a microbiology laboratory, it isadvisable to choose the outer dimensions of the mask such that theliquid removed, together with any microorganisms which may possiblyremain suspended in it, cannot penetrate to the outer edge or top of themask, even when the liquid is completely absorbed, in order that themask can still be gripped and moved without any risk of contamination.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be better understood by referring to the followingillustrations. The elements in the illustrations are not necessarily toscale, but are intended primarily to illustrate the principles of theinvention (mainly schematically). In the illustrations, the samereference numbers designate corresponding elements in the differentviews.

FIG. 1 is a schematic diagram of an example embodiment for a mask of anabsorbent material with 96 holes (arranged in 8 rows and 12 columns).

FIGS. 2A-2C give a schematic illustration of an embodiment of themethods.

FIG. 3 is a schematic diagram of an example embodiment for a mask of anabsorbent material with indentations instead of holes.

FIGS. 4A and 4B provide a schematic illustration of the use of a maskwith corresponding frame or vertical guide for the purpose of alignmentand guidance.

FIGS. 5A-5C present another schematic illustration of a furtherembodiment of the methods.

DETAILED DESCRIPTION

While the invention has been illustrated and explained with reference toa number of different embodiments, those skilled in the art willrecognize that various changes in form and detail may be made to itwithout departing from the scope of the technical teaching as defined inthe appended claims.

FIG. 1 depicts a plan view of a mask of rectangular form (10), which hasan array of 96 holes (12) arranged in a nine-millimeter grid in anarrangement of eight rows by twelve columns. Smaller (e.g. 48) or larger(e.g. 384) arrays are also conceivable, however. The array can generallycorrespond to the arrangement of sample spots on a conventional,standardized sample support for ionization by means of matrix assistedlaser desorption (MALDI). The dimensions of the mask (10) can be 127.76mm (length)×85.48 mm (width), corresponding to a microtitration plate,with a thickness of around two to five millimeters.

FIG. 2A shows an arrangement of equal droplets (14) in a row of eight ona flat substrate which can correspond to a MALDI sample support plate(16). A mask (10) of an absorbent material, which has an array of holes(12) arranged opposite all the droplets (14), is positioned above theflat substrate. Each sample spot on the support plate (16) thus has ahole opposite it (12). The mask (10) is moved slowly towards thesubstrate, whereby the droplets (14) come into contact with theabsorbent material at the hole edges once the mask has been lowered to acertain point so that the liquid is removed from the droplets (14)laterally via capillary forces, FIG. 2B.

This lowering movement may end when the mask (10) is lying on the flatsubstrate, as depicted; it is also possible to keep the mask (10)slightly above the substrate without coming into contact. This canprevent a lateral spread of droplet liquid in the gap between mask (10)and substrate, which could lead to the mutual contamination of theindividual droplets (14). To ensure that each droplet (14), even when itis applied slightly asymmetrically or does not cover the whole samplespot, comes into contact with the absorbent material of the mask (10),the mask (10) can be moved laterally to and fro slightly, as indicatedby the double-headed arrow (18).

The lateral absorption of the liquid from the droplet, starting from themiddle of the droplet, is completed in a very short time, usually a fewseconds up to around one minute at the most. Afterwards, the mask (10)can be lifted again and removed, FIG. 2C. The liquid removed is safelyheld in the capillary matrix of the mask (10), so there is no dangerthat it will drop out again as it is being lifted and contaminate theflat substrate. On the contrary, it is a very safe and reliable way toremove the liquid. The partially saturated mask (10) is typicallydisposed of as a consumable, which is advantageous particularly forapplications in microbiology. It could also be washable and thenre-usable where appropriate, however.

Sedimented material, such as microorganisms, which is enclosed in thedroplets (14) is not removed when the liquid is gently drawn off withthe aid of capillary forces. It does not come into contact with theedges of the holes (12) (or indentations), but remains in the center ofthe spot on the surface of the flat substrate on which the droplets (14)were deposited. The sedimented material, now largely free of liquid, isthus available for further processing such as sample preparation forionization by means of matrix assisted laser desorption or similarprocess steps.

FIG. 3 shows a schematic side view of a row of eight indentations whichhave, for example, been pressed into a stiff and rigid mask fabric suchas non-woven material. When the indentations are designed appropriatelyand adapted to the expected droplet shape, the liquid can be made tocome into contact not only with the edge (as with the hole version) butalso with the bottom of the indentation, or at least parts of theindentation surface, which may further accelerate the removal process.Since the droplets are not visible through the mask in this embodiment,care must be taken that the mask is aligned correctly—particularly whenthis is done manually—with the array of droplets on the flat substrate.

In a preferred embodiment, the dimensions of the strips between theindentations or holes, compared to the spacing of the indentations orholes themselves, are chosen such that the liquids absorbed from thedifferent individual droplets do not run into each other, thuspreventing cross-contamination. Furthermore, the thickness of the maskand the width of the side edge are preferably dimensioned so that theliquid is not drawn right to the top or the edges. If we assumecylindrical droplets with a volume πr²×h as our model (r=droplet radius;h=droplet height), which is drawn into a cylindrical ring around a hole,which for simplicity has the same volume 2πr×dr×h, then the ratio ofring width dr to droplet radius is given by dr/r=0.5. This means thatthere is no mutual penetration of liquids of neighboring individualdroplets when the width of the strips is given by: s>2×dr=2×0.5×r=r.According to this simple model, the strip width is therefore preferablychosen to be larger than half the hole diameter (or indentationdiameter). Similar considerations can be applied to the mask edge andthe mask thickness.

To make the mask (10) easier to handle, it can be inserted or clampedinto a frame (20). The frame (20) can be dimensioned so as to create aflush fit around a sample support (16) which contains the array ofdroplets, for example, as depicted in FIG. 4A. It can be designed as adisposable article, which is disposed of together with the saturatedmask (10), or can be washable and re-usable. Possible designs encompassa frame (20) with stepped inner contour, on which the mask (10) can beplaced with friction locking. If the frame (20) slides down around theouter sample support contour, as depicted, contact with the liquid isestablished below a certain point. The frame (20) has furthermore theadvantage that it provides reliable alignment and guidance of the arrayof holes relative to the array of droplets. If the inner contour of theframe (20) and the outer contour of the sample support (16) are notdimensioned so as to be completely flush, but have a certain amount ofplay, a slight lateral movement can be executed to guarantee that theliquid of all droplets comes into contact with the mask.

In an alternative embodiment, the frame can be fixed to the mask. Aprojecting, custom-cut edge of the absorbent material can be folded andthen impregnated with a plastic material, for example, which then setsto ensure stability and rigidity (monolithic version). The frame can, ifappropriate, also be attached to the outer circumference of the maskusing an injection molded plastic.

In a version sketched in FIG. 4B, a sample support (16), as a flatsubstrate which supports the array of droplets, can be inserted into avertical guide (22) which surrounds it on all sides. The mask (10) canthen have similar dimensions to the sample support (16) and slide slowlydownwards onto the sample support (16) from the top opening of thevertical guide. Grip recesses in the walls of the vertical guide (notshown here) can facilitate the insertion and removal of sample support(16) and mask (10).

In another implementation of the principles set out herein, FIG. 5Ashows again an arrangement of equal droplets (14) in a row of eight on aflat substrate which can correspond to a MALDI sample support (16),similar to FIG. 2A. A rigid, stiff plate (24) of an absorbent material,without any profile, is positioned above the flat substrate. The plate(24) is moved slowly towards the substrate, whereby the protruding partsof the droplets (14) come into contact with the absorbent material oncethe plate (24) has been lowered to come to rest on the spacer ridge(26), which is located laterally at a receptacle (28) accommodating theflat substrate. The distance above the flat substrate surface kept bythe spacer ridge (26) can amount to about one third to about half thedroplet diameter, for example. The peripheral contact facilitates theremoval of liquid from the droplets (14) via capillary forces, FIG. 5B.No special alignment of plate (24) and droplets (14) is necessary inthis variant.

Instead of locating the spacer ridge at a receptacle (28), it could alsobe mounted laterally on the surface of the rigid, stiff plate (24)facing the flat substrate itself, as indicated by the dotted contour.This alternative design affords better adaptability to different dropletsizes, in particular when the plate (24) is designed as a consumable. Toaccelerate the aspiration of the liquid into the absorbent material ofthe rigid, stiff plate (24) at the points of contact, the plate (24) canbe moved laterally to and fro slightly, as indicated by thedouble-headed arrow (18).

As expounded before, the absorption of the liquid from the droplets (14)is completed in a very short time, usually a few seconds up to aroundone minute at the most. Afterwards, the partly saturated plate (24) canbe lifted again and removed, FIG. 5C. The liquid removed is safely heldin the capillary matrix of the plate (24), so there is no danger that itwill drop out again as it is being lifted and contaminate the flatsubstrate. On the contrary, it is a very safe and reliable way to removethe liquid.

Sedimented material, such as microorganisms, which is enclosed in thedroplets (14) is not removed when the liquid is gently drawn off withthe aid of capillary forces. By virtue of the spacer ridge (26) whichkeeps the surface of the rigid, stiff plate (24) at a distance, forexample about one third to about half a droplet diameter above the flatsubstrate, it does not come into contact with the absorbent material ofthe plate (24) at all, but remains in the center of the spot on thesurface of the flat substrate on which the droplets (14) were deposited.The sedimented material, now largely free of liquid, is thus availablefor further processing such as sample preparation for ionization bymeans of matrix assisted laser desorption or similar process steps, ashas been explained before.

Further embodiments of the invention are conceivable in addition to thedesigns described by way of example. With knowledge of this disclosure,the person skilled in the art is easily able to design further,advantageous sample processing methods for infrared spectroscopic ormass spectrometric measurement using a desorbing ionization method,which are to be contained in the scope of protection of the claims,including any possible equivalents as the case may be.

The invention claimed is:
 1. A method for drawing-off liquid fromindividual droplets which are in an arrangement on sample spots of aflat substrate and contain sedimented material, the method comprising:positioning a mask of an absorbent material above the flat substrate,the mask comprising a pattern of indentations or holes that correspondsto the arrangement of the individual droplets in such a way that eachsample spot has a respective one of said indentations or holes oppositeit; and lowering the mask so that edges of the indentations or holescome into contact with peripheral parts of the individual droplets suchthat liquid is drawn off into the absorbent material.
 2. The methodaccording to claim 1, wherein a metal or ceramic plate is used as theflat substrate.
 3. The method according to claim 1, wherein 48, 96, 384or 1536 of said individual droplets are arranged in a regular pattern onthe flat substrate.
 4. The method according to claim 1, wherein thesedimented material enclosed in the individual droplets containsmicroorganisms.
 5. The method according to claim 1, wherein the volumeof any one of said droplets is approximately between one and twelvemicroliters.
 6. The method according to claim 1, wherein the mask andthe flat substrate are moved laterally relative to each other to ensurethat all of said individual droplets come into contact with thecorresponding indentations or hole edges.
 7. The method according toclaim 1, wherein each of said sample spots comprises a hydrophilic areaon which is located one of said droplets, a size of each hydrophilicarea limiting a maximum diameter of a droplet located thereupon to beinglarger than a diameter of a respective one of said indentations or holesthat is opposite it.
 8. The method according to claim 1, wherein theindividual droplets comprise at least one of nutrient liquid and washingliquid.